Dimeric association of Escherichia coli RNA polymerase alpha subunits, studied by cleavage of single-cysteine alpha subunits conjugated to iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate.
Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant alpha subunits were conjugated with the cutting reagent iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within approximately 12 A of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.
Miyake
R
R
Department of Chemistry, University of California, Davis 95616, USA.
Murakami
K
K
Owens
J T
JT
Greiner
D P
DP
Ozoline
O N
ON
Ishihama
A
A
Meares
C F
CF
D000469:Alkaline Phosphatase; D019921:Biotinylation; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007502:Iron Chelating Agents / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds / Q000378:metabolism*; D017510:Protein Folding; D019809:Streptavidin
D007502:Iron Chelating Agents; D009942:Organometallic Compounds; C042007:1-(4-bromoacetamidobenzyl)EDTA; D019809:Streptavidin; D004492:Edetic Acid; D012321:DNA-Directed RNA Polymerases; D000469:Alkaline Phosphatase; D003545:Cysteine